RESUMO
BACKGROUND: The delivery of a complete genomic DNA locus in vivo may prove advantageous for complementation gene therapy, especially when physiological regulation of gene expression is desirable. Hydrodynamic tail vein injection has been shown to be a highly efficient means of non-viral delivery of plasmid DNA to the liver. Here, we apply hydrodynamic tail vein injection to deliver and express large genomic DNA inserts > 100 kb in vivo. METHODS: Firstly, a size series (12-172 kb) of bacterial artificial chromosome (BAC) plasmids, carrying human genomic DNA inserts, episomal retention elements, and the enhanced green fluorescent protein (EGFP) reporter gene, was delivered to mice by hydrodynamic tail vein injection. Secondly, an episomal BAC vector carrying the whole genomic DNA locus of the human low-density lipoprotein receptor (LDLR) gene, and an expression cassette for the LacZ reporter gene, was delivered by the same method. RESULTS: We show that the efficiency of delivery is independent of vector size, when an equal number of plasmid molecules are used. We also show, by LacZ reporter gene analysis, that BAC delivery within the liver is widespread. Finally, BAC-end PCR, RT-PCR and immunohistochemistry demonstrate plasmid retention and long-term expression (4 months) of human LDLR in transfected hepatocytes. CONCLUSION: This is the first demonstration of somatic delivery and long-term expression of a genomic DNA transgene > 100 kb in vivo and shows that hydrodynamic tail vein injection can be used to deliver and express large genomic DNA transgenes in the liver.
Assuntos
Pareamento de Bases/genética , DNA/metabolismo , Técnicas de Transferência de Genes , Genoma Humano/genética , Receptores de LDL/genética , Animais , Cromossomos Artificiais Bacterianos/genética , Feminino , Vetores Genéticos , Humanos , Injeções , Fígado/citologia , Fígado/metabolismo , Camundongos , Plasmídeos/metabolismo , Fatores de Tempo , Transgenes , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Vein graft stenosis is a common problem after bypass surgery. Vein grafts are ideal targets for gene therapy because transduction can be made ex vivo before grafting. Since chemokines and inflammatory factors are involved in vein graft thickening, we tested a hypothesis that the vaccinia virus anti-inflammatory protein 35K which can sequester CC-chemokines, can reduce vein graft thickening in vivo. MATERIALS AND METHODS: We used adenovirus-mediated gene transfer (1x10(9) pfu/ml) of 35K and compared its effects on reducing stenosis in a rabbit jugular vein graft model with tissue inhibitor of metalloproteinase-1 (TIMP-1) and LacZ control gene. TIMP-1 was used in this study because it has previously been shown to inhibit vein graft stenosis in other model systems. The expression of transgenes in the transduced segments was confirmed by RT-PCR. Vein grafts were analyzed using immunohistological and morphometric methods at the three-day time-point and at two-week and four-week time-points. RESULTS: It was found that the anti-inflammatory protein 35K was an efficient factor in reducing neointima formation at the two-week time-point, indicating that inflammatory factors play an important role in vein graft stenosis. At the four-week time-point, 35K still showed a reduced accumulation of macrophages. TIMP-1 also tended to reduce neointimal thickening at the two-week time-point as compared to LacZ. CONCLUSION: It was found that 35K is an efficient factor in reducing neointima formation, macrophage accumulation and proliferation in rabbit vein grafts after adenoviral ex vivo gene transfer.
